Ultrastructural Organization of Recombinant Marburg Virus Nucleoprotein: Comparison with Marburg Virus Inclusions

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JOURNAL OF VIROLOGY, Apr. 2000, p. 3899–3904 Vol. 74, No. 8
0022-538X/00/$04.00⫹0
Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Ultrastructural Organization of Recombinant Marburg Virus
Nucleoprotein: Comparison with Marburg Virus Inclusions
LARISSA KOLESNIKOVA,1 ELKE MÜHLBERGER,2 ELENA RYABCHIKOVA,1
2
AND STEPHAN BECKER *

State Scientific Research Center of Virology and Biothechnology, Vector Institute of Molecular Biology,
Laboratory of Ultrastructure and Pathomorphology, 633159 Koltsovo, Novosibirsk Region, Russia,1
and Institut für Virologie der Philipps-Universität Marburg, D-35037 Marburg, Germany2
Received 30 August 1999/Accepted 20 January 2000

HeLa cells expressing the recombinant Marburg virus (MBGV) nucleoprotein (NP) have been studied by

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immunoelectron microscopy. It was found that MBGV NPs assembled into large aggregates which were in close
association with membranes of the rough endoplasmic reticulum. Further analysis of these aggregates revealed
that NPs formed tubule-like structures which were arranged in a hexagonal pattern. A similar pattern of
preformed nucleocapsids was detected in intracellular inclusions induced by MBGV infection. Our data
indicated that MBGV NP is able to form nucleocapsid-like structures in the absence of the authentic viral
genome and other nucleocapsid-associated proteins.

Marburg virus (MBGV) and Ebola virus (EBOV) make up structural proteins (5, 8). The nucleocapsid of the virion is
the family of Filoviridae, which belongs to the order Monon- composed of the viral RNA and four proteins, the nucleopro-
egavirales. MBGV causes a severe hemorrhagic disease in hu- tein (NP), P (formerly called VP35), the viral protein VP30,
mans and nonhuman primates (22). The recent outbreak of and the catalytic subunit of the polymerase (L). Two putative
MBGV hemorrhagic fever in the Democratic Republic of the matrix proteins, VP24 and VP40, are located between the
Congo underlines the emerging potential of this pathogen (25). nucleocapsid and the envelope, which is decorated with the
MBGV is an enveloped virus with a nonsegmented negative- surface protein (GP) (2, 3, 7, 13, 15, 20).
strand RNA genome 19.1 kb in length, which encodes seven Viral reproduction takes place in the cytoplasm, and the

FIG. 1. IEM analysis of ultrathin section of MVA-T7-infected HeLa cells expressing recombinant NP. Immunogold labeling was carried out using a monoclonal
antibody directed against MBGV NP (dilution, 1:10) and a goat anti-mouse antibody conjugated with colloidal gold (bead diameter, 5 nm) (dilution, 1:50). NP
aggregates appeared as two long sheets of a reticular network of electron-dense material heavily labeled with gold particles. The NP aggregates are in close association
with the membrane of the rER. Bar, 150 nm.

* Corresponding author. Mailing address: Institut für Virologie der
Philipps-Universität Marburg, Robert-Koch-Strasse 17, D-35037 Mar-
burg, Germany. Phone: 06421-28-5433. Fax: 06421-28-5482. E-mail:
becker@mailer.uni-marburg.de.

3899

3900 NOTES J. VIROL.

infectious particles are released by budding at the cell surface
(10, 11, 16–19). MBGV morphogenesis commences with the
formation of intracytoplasmic blurred wavy strands which are
first arranged in loose accumulations and then scattered (17).
More frequently, MBGV inclusions appear as amorphous elec-
tron-dense matrices or sheets. It has been observed that
MBGV inclusions show the manifestation of nucleocapsid for-
mation only shortly before budding, in contrast to EBOV in-
clusions, which as a rule contain easily discernible preformed
nucleocapsids (11). Sometimes, sections of MBGV inclusions
exhibit thin-walled tubule-like structures (TLS) which are or-
ganized in a hexagonal pattern (11, 17). However, the exact
protein composition of these TLS has not been identified.
Immunoelectron microscopic (IEM) analysis revealed that
MBGV inclusions contain a large amount of NP, but the in-

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ternal arrangement of NP has been unknown until now (10,
11).
Encapsidation of the genomic RNA by the major nucleo-
capsid protein (NP) is presumed to be the first step in the
assembly process of MBGV nucleocapsid (1, 19). The mecha-
nism of this initial step is still unknown. Studies with other
viruses of the order Mononegavirales showed that nucleopro-
teins are able to assemble into nucleocapsid-like structures
independently of other viral proteins (4, 9, 12, 14, 23). Since
the protein composition of the nucleocapsid complexes of Filo-
viridae is different from that of the other Mononegavirales (four
instead of three nucleocapsid proteins [1]), it is unclear wheth-
er NP also contains the information for the assembly of nu-
cleocapsid-like structures. Among the proteins of the MBGV
nucleocapsid complex, only NP has the potential to self-assem-
ble into large aggregates when it is expressed in the absence of
other viral proteins (3). However, features of NP packaging
inside the aggregates have not yet been investigated.
To examine whether the NP is able to self-assemble into
nucleocapsid-like structures, we have analyzed the ultrastruc-
tural organization of aggregates formed by the recombinant
NP and compared the results with the ultrastructural organi-
zation of viral inclusions in MBGV-infected cells. We report
here that recombinant MBGV NP assembles into TLS ar-
ranged in a hexagonal pattern. The organization and morphol-
ogy of TLS in recombinant NP aggregates are very similar to
those of the preformed nucleocapsids which we identified in
viral inclusions in MBGV-infected cells.
For NP expression, approximately 107 HeLa cells were in-
fected with recombinant vaccinia virus MVA-T7 (24) and sub-
sequently transfected with 1 ␮g of pT/NP as described previ-
ously (15). At 8 h postinfection (hpi), cells were washed with
Hanks balanced salt solution (HBS), fixed with HBS contain-
ing 2.5% paraformaldehyde and 0.1% glutaraldehyde, dehy-
drated, and embedded in acrylic resin (LR gold; Sigma). Po-
lymerization was carried out at ⫺20°C by UV irradiation. IEM
was performed according to the method of Geisbert and Jahr-
ling (11) by using a monoclonal antibody against NP, diluted
10-fold. The bound antibodies were detected with a goat anti-
mouse antiserum conjugated with colloidal gold beads (diam-
eter, 5 nm). FIG. 2. Electron microscopical analysis of ultrathin sections of MBGV-in-
fected Vero cells at 43 hpi. (a) Morphological variability of MBGV inclusions.
For comparative electron microscopical studies, Vero cells MBGV inclusions appeared as thin strands (small arrows) or as thick sheets with
were infected with MBGV strain Musoke at a multiplicity of longitudinal striation (large arrows). The thin strands of MBGV inclusions are in
infection of 1 PFU per cell and harvested at 43 hpi. Cells close association with the outer membrane of the nuclear envelope and with a
were fixed with HBS containing 4% paraformaldehyde, smooth membrane structure (arrowheads). N, nucleus. Bar, 200 nm. (b) Large
sheets of MBGV inclusions. Cross-sectioned TLS in MBGV inclusions, which
postfixed with HBS containing 1% osmium tetraoxide, dehy- represent preformed nucleocapsids, display circles in a regular arrangement
drated, and embedded in Epon and Araldite. Ultrathin sec- (large arrow). Longitudinally sectioned thin-walled TLS surrounded by more-
tions were stained with uranyl acetate and lead citrate (19). electron-dense matrices (mature nucleocapsids) are seen inside and outside the
The gold particles and structures of infected cells were visual- large MBGV inclusion (small arrows). The association of MBGV inclusions with
an rER membrane is indicated by arrowheads. Bar, 150 nm.
ized using a Hitachi 600 electron microscope. Measurements
were made from prints at a final magnification of 200,000.

VOL. 74, 2000 NOTES 3901

Numerical values are presented as means ⫾ standard devia-
tions (n ⫽ 20). Student’s t test was used for statistical analysis
(P ⬍ 0.01).
IEM analysis of HeLa cells expressing NP revealed electron-
dense inclusions within the cytoplasms, which were heavily
labeled with gold particles (Fig. 1). Areas of the cytoplasm
which were devoid of NP inclusions did not show gold labeling.
As a consequence of the vaccinia virus infection, viral factories,
immature viral particles, and mature viral particles were ob-
served. The used antibody did not cross-react with vaccinia
virus-specific structures. Furthermore, background staining of
uninfected cells was not detectable (data not shown).
The NP aggregates appeared as a reticular network of elec-
tron-dense material interspersed with electron-lucent material.
The NP aggregates were detected mostly in close association

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with rough endoplasmic reticulum (rER) membranes and
sometimes with the outer membrane of the nuclear envelope.
The NP aggregates formed long sheets with one end in close
association with rER channels or were localized between rER
channels (Fig. 1). It is assumed that the NP-induced sheets
were formed because nascent NP peptide chains were attached
to the preexisting NP molecules, thus preventing diffuse spread-
ing throughout the cytoplasm.
To investigate whether intracytoplasmic MBGV-specific in-
clusions are also associated with the membrane of the rER, we
have studied ultrathin sections of MBGV-infected Vero cells
harvested at 43 hpi. Association of intracytoplasmic MBGV
inclusions with rER membranes did not appear as frequently
as with aggregates of recombinant NP. However, in several
cases, an association between rER membranes and both small
wavy strands (Fig. 2a) and large sheets of intracytoplasmic
MBGV inclusions was observed (Fig. 2b). The differences be-
tween MBGV inclusions and NP aggregates with regard to
their rER associations have yet to be explained.
Detailed analysis of aggregates formed by recombinant NP
in HeLa cells revealed in some sections gold-labeled regular
striation or regularly arranged circles (Fig. 3). The periodicity
within the striated patterns was approximately 25 nm. In some
areas of longitudinal sections the gold particles were arranged
in rows (Fig. 3a, inset). The circular domains were hexagonally
organized (Fig. 3b). The inner diameters of the circles were
18.5 ⫾ 0.4 nm, and the outer diameters were 27.9 ⫾ 0.4 nm.
The distances between the circles were 16.1 ⫾ 0.6 nm, and the
distances between the centers of the circles were 44.0 ⫾ 0.5
nm. As individual circular domains were labeled with up to six
gold particles delineating their perimeters (Fig. 3b, inset) it
became clear that these circular domains are comprised of NP
molecules.
Based on the maximum number of antibodies binding to one
of the ring-like structures, it is presumed that these structures
are formed by at least six subunits which are composed of NP.
However, it cannot be ruled out that the subunits are com-
posed of a multitude of NP molecules, e.g., six dimers, trimers,
or even tetramers. Peters et al. (17) described in a study per-
formed with transmission electron microscopy that one cross-
sectioned nucleocapsid in MBGV inclusions is composed of 24

FIG. 3. IEM analysis of ultrathin sections of aggregates formed by recombi-
nant NP. Immunogold labeling was performed as described in the legend to Fig.
1. (a) NP aggregate labeled with gold showing striated matrices with a periodicity
of approximately 25 nm. The inset shows a row of gold particles which occurred
in a longitudinal section. Bar, 100 nm. (b) NP aggregates labeled with gold
showing a hexagonal pattern (hexagons are indicated by squares and crosses).
Bar, 100 nm. The inset shows one circular domain labeled by five gold particles,
one of which is slightly dislocated.

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   FIG. 4. Comparative analysis of ultrathin sections of viral inclusions in MBGV-infected cells and aggregates formed by recombinant NP. (a) Cross-section of viral
inclusion in MBGV-infected Vero cells at 43 hpi. Preformed nucleocapsids are arranged in a hexagonal pattern (white squares) and embedded in electron-dense
matrices. Some of the thin-walled TLS in the periphery of the inclusion are surrounded by more-electron-dense matrices and represent mature nucleocapsids (arrows).
Inset 1 (fragment of the hexagonal pattern which is marked by white squares) shows thin-walled TLS surrounded by a narrow electron-translucent region (arrow). Inset
2 (fragment of the viral inclusion in panel a [one of the marked mature nucleocapsids]) represents thin-walled TLS surrounded by a region of high electron density.
Inset 3 shows a cross-section of an MBGV particle. The thicknesses and electron densities of the regions surrounding the mature nucleocapsid within the MBGV
inclusion and within the viral particle are almost identical. Bar, 25 nm. Inset 4 shows a cross-sectioned TLS formed by recombinant NP (the fragment of Fig. 3b which
is marked by black squares). The photo of inset 4 is printed at the same magnification as the photo of the MBGV inclusion. Bar, 70 nm. (b and c) Schematic illustrations
of cross-sectioned inclusions induced either by recombinant NP infection (b) or by MBGV infection (c). Hexagonally arranged black circles represent TLS inside an
NP aggregate (b) and preformed nucleocapsids inside an MBGV inclusion (c). MBGV inclusions (c) contain preformed nucleocapsids embedded in electron-dense
matrices (shown by shading) and mature nucleocapsids which are characterized by their highly electron-dense appearance (shown by superimposed circular shading).

                                                                                 3902

VOL. 74, 2000 NOTES 3903

grains with diameters of less than 5 nm. The helical nucleo- gregates composed of recombinant NP further revealed that
capsid of Sendai virus contains 13 NP subunits in each turn (6). the hexagonal organization of the nucleocapsids is related to
The varied patterns of NP aggregates in ultrathin sections that of NP. For illustration, the arrangement of the nucleocap-
were related to different planes of the sections. Thus, the pres- sid-like structures inside the inclusions, either induced by re-
ence of striated or hexagonal patterns or a reticular network combinant NP or by MBGV infection, is schematically drawn
suggested that NP forms TLS which are connected to each in Fig. 4b and c. It is shown that the overall organization of
other. The regularly arranged TLS in NP aggregates that have thin-walled TLS formed by recombinant NP (Fig. 4a, inset 4,
a striated appearance in a longitudinal section appear as hexa- and b) is conserved in MBGV-induced inclusions (Fig. 4a and
gons in cross-sections and exhibit a reticular network in a c), with the difference that viral inclusions additionally con-
skewed section. It is noteworthy that in some sections of NP tained electron-dense material inside and surrounding the TLS
aggregates all three kinds of appearances of TLS could be that were not detected in aggregates of recombinant NP. It
observed, indicating that these aggregates are curved (not cannot yet be ruled out that the interaction between the TLS is
shown). This result might reflect a certain flexibility of TLS. made by cellular proteins. In any case, it seems very likely that
It has been shown previously that recombinant nucleopro- NP is a scaffold for viral assembly. It is interesting that the
teins of Paramyxoviridae form helices (4, 9, 14, 23) and that hexagonal arrangement of preformed nucleocapsids is also ob-

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recombinant nucleoproteins of rabies virus can form both he- served in EBOV inclusions. Other families of the order
lices and rings, depending on conditions of purification (12). Mononegavirales, however, display viral inclusions with irregu-
Analysis of postnuclear supernatant of HeLa cells expressing larly arranged nucleocapsids (21; our unpublished observa-
MBGV NP, fractionated on sucrose gradients (5 to 30%), tions). Self-assembly of nucleoproteins into nucleocapsid-like
revealed filamentous structures or extremely elongated helices. structures has been detected with the families Paramyxoviridae
The rings detected with rabies virus were not detected (data (4, 9, 14, 23) and Rhabdoviridae (12). Thus, the ability of
not shown). This result could be explained by the extreme nucleoproteins to determine the nucleocapsid structure ap-
fragility of MBGV nucleocapsids, which might prevent preser- pears to be a general pattern among the order Mononegavi-
vation of the original structure during the isolation procedure rales.
(1). Although our results rather suggested a helical arrange- In this report we demonstrated for the first time that (i)
ment of TLS, detailed analysis has to be carried out before MBGV NP assembles into TLS arranged in a hexagonal pat-
definite statements on the three-dimensional packaging of NP tern in the absence of the authentic viral genome and the other
inside the TLS can be made. nucleocapsid-associated proteins, (ii) TLS in NP aggregates
Viral inclusions in MBGV-infected cells were analyzed to are curved, (iii) there is a close association between NP aggre-
compare the organization of these structures with that of ag- gates and membranes of the rER, and (iv) the organization and
gregates of recombinant NP. A remarkable morphological morphology of TLS in recombinant NP aggregates are very
variability of intracytoplasmic MBGV inclusions was detected, similar to those of the preformed nucleocapsids which were
as has also been shown in previous reports (10, 11, 16–19) (Fig. identified in inclusions of MBGV-infected cells.
2). Depending on the plane of the section, MBGV inclusions
exhibited either hexagonally arranged thin-walled circles (Fig. We thank Andreas Holzenburg, University of Leeds, for helpful
4) or a striated pattern (Fig. 2), suggesting the presence of TLS discussion, critical reading of the manuscript, and encouragement. We
as in NP aggregates. In some sections of MBGV inclusions, also thank Brigitte Agricola and Claudia Schmidt (Institute of Cell
both striation and circles could be observed, suggesting that Biology, Marburg, Germany), Sonja Heck (Institute of Virology, Mar-
TLS in MBGV inclusions, like the TLS formed by recombinant burg, Germany), and Marina Vorobjova (SRC VB Vector Novosibirsk,
NP, were rather curved (not shown). The inner diameters of Russia) for assisting with ultrastructural analysis. In addition, we thank
the thin-walled tubules in MBGV inclusions were 17.6 ⫾ 0.3 Michael Weik for preparing the artwork.
This work was supported by the European Union (grant INTAS
nm, and the outer diameters were 26.3 ⫾ 0.4 nm. The distances 96-1361) and by the Deutsche Forschungsgemeinschaft (grants SFB
between the thin-walled tubules were 18.2 ⫾ 0.5 nm, and the 286, TPA6).
distances between the centers of TLS in MBGV inclusions
were 44.5 ⫾ 0.6 nm. Thus, thin-walled TLS in MGBV inclu-
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